Methods for Altering One or More Parameters of a Measurement System

ABSTRACT

Methods for altering one or more parameters of a measurement system are provided. One method includes analyzing a sample using the system to generate values from classification channels of the system for a population of particles in the sample. The method also includes identifying a region in a classification space in which the values for the populations are located. In addition, the method includes determining an optimized classification region for the population using one or more properties of the region. The optimized classification region contains a predetermined percentage of the values for the population. The optimized classification region is used for classification of particles in additional samples.

PRIORITY CLAIM

The present application is a continuation from prior U.S. applicationSer. No. 11/031,905 filed Jan. 7, 2005 which claims priority to U.S.Provisional Application No. 60/536,466 filed Jan. 14, 2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to methods for altering one or moreparameters of a measurement system. Certain embodiments relate tomethods and systems for optimizing one or more parameters of ameasurement system for classifying particles of a population.

2. Description of the Related Art

The following descriptions and examples are not admitted to be prior artby virtue of their inclusion within this section.

Generally, flow cytometers provide measurements of fluorescenceintensity of laser excited polystyrene beads as they pass linearlythrough a flow chamber. In some systems, there are four measurementsthat are performed: the level of light scattered by a bead at 90 degreesto the excitation source, two measurements of fluorescence used todetermine the bead “identity” or “classification,” and a thirdfluorescence measurement typically used to detect and/or quantify asurface chemical reaction of interest. Each of the three fluorescentmeasurements is made at a different wavelength. These and any otherfluorescent measurements are performed by different “channels” of thesystem (e.g., reporter channels, classification channels), which includea detector and possibly other components (e.g., optical components,electronic components, etc.) coupled to the detector.

In one example, the fluorescence measurement of the chemical reaction isquantified by optically projecting an image of the bead as it passesthrough the illumination zone of the excitation laser on thephotosensitive area of a photomultiplier tube (PMT). The output of thePMT is a current pulse, which is then conditioned by analog electronicsand digitized by an analog to digital (A/D) converter. The resultantdigital values obtained from the A/D converter may be furtherconditioned in the digital domain by a digital signal processing (DSP)algorithm. The end product per bead is a single integer value, which isgenerally proportional to the chemical reaction on the surface of thebead.

Each flow cytometer based system such as the Luminex 100 system, whichis manufactured by Luminex Corp., Austin, Tex., displays results ofparticle measurements (e.g., the 100-region LabMAP) in a slightlydifferent way than a “typical” flow cytometer instrument. Thesedifferences in the displays are a result of the accumulated tolerancefor many of the components of the system such as the diode laser, thephotodiodes, the optical filters, and the electronics used to processthe data. In particular, particles are classified by comparing valuesgenerated during analysis of the particles to regions located in aclassification space (e.g., the Map). Particles having values that arelocated within a region in the classification space are assigned theclassification corresponding to this region. Therefore, in order toaccount for the accumulated tolerance of the system described above, thesize of the regions in the classification space that are used toclassify different populations are made larger than necessary to containthe values of the different populations.

One result of using these larger than necessary classification regionsis inconsistent classification of particle populations between onesystem and another. For instance, one system might be able to classify95% of a particle population as belonging to a particular region, and0.5% of that population would typically be misclassified in anotherregion, whereas a different system might correctly classify 98% of thisparticle population and misclassify a smaller percentage of thepopulation. Therefore, using a larger than necessary classificationregion results in poor system-to-system matching. System-to-systemmatching, however, may be desirable, for example, when multiplemeasurement systems are used in a single facility or organization toperform assays on biological samples. In this manner, results obtainedusing one measurement system may be directly compared to resultsobtained using a different measurement system.

Obviously, one way to reduce the size of the classification regions isto reduce the accumulated tolerance of the system. One way to reduce theaccumulated tolerance of the system is to manufacture the system usingcomponents that have extremely narrow tolerances. However, using suchcomponents places a significant burden on manufacturing personnel insourcing these components. In addition, rigorous assembly efforts can beused to try to compensate for tight tolerances. However, like theextremely narrow tolerance components, using rigorous assembly effortsincreases the complexity and difficulty of manufacturing. Therefore,currently available methods for reducing the size of the classificationregions increase system manufacturing time, decrease manufacturingthroughput, and increase overall system cost.

Accordingly, it would be desirable to reduce the size of theclassification regions such that the system can classify particles withgreater system accuracy and greater system-to-system uniformity withoutcomplicating the manufacturing process, increasing the manufacturingtime, decreasing manufacturing throughput, and increasing overall systemcost.

SUMMARY OF THE INVENTION

The following description of various embodiments of methods for alteringone or more parameters of a measurement system is not to be construed inany way as limiting the subject matter of the appended claims.

One embodiment of the invention relates to a method for altering one ormore parameters of a measurement system. The method includes analyzing asample using the system to generate values from classification channelsof the system for a population of particles in the sample. The methodalso includes identifying a region in a classification space in whichthe values for the population are located. In addition, the methodincludes determining an optimized classification region for thepopulation using one or more properties of the region. The optimizedclassification region contains a predetermined percentage of the valuesfor the population. The optimized classification region can be used forclassification of particles in additional samples.

In some embodiments, the optimized classification region has one or moreproperties that are different from the one or more properties of theregion. The one or more properties include size, shape, position, orsome combination thereof. In one embodiment, the method includescalibrating the system prior to the analyzing step. The sample mayinclude, in some embodiments, a Map Calibration Reagent. The values maybe expressed in linear units or logarithmic units.

In an embodiment, the one or more properties of the region include anaverage, mean, peak, or median of the values in the region and astandard deviation of the values in the region. In another embodiment,the optimized classification region centers on a median of the values inthe region plus a number of standard deviations away from the median. Inan additional embodiment, the optimized classification region is definedby a predetermined sized boundary surrounding a median of the values forthe population. The size of the optimized classification region is, insome embodiments, a minimum size that contains the predeterminedpercentage of the values for the population.

In one embodiment, the values from the classification channels includefluorescence values. In a different embodiment, the values from theclassification channels include light scatter intensity values. In otherembodiments, the values from the classification channels include volumemeasurements of the particles. In another embodiment, the values mayinclude some combination of the different values (e.g., fluorescencevalues and light scatter intensity values, etc.).

The sample may, in some embodiments, include one or more additionalpopulations of particles. In one such embodiment, the method isperformed for the one or more additional populations. In this manner, anoptimized classification region may be determined for each of the one ormore additional populations. In another such embodiment, the method mayinclude interpolating an optimized classification region for anotherpopulation of particles that was not included in the sample using theoptimized classification regions of the population and the one or moreadditional populations.

In one embodiment, the one or more properties of the region may include,as described above, an average, mean, peak, or median of the values forthe population and a standard deviation of the values for thepopulation. In one such embodiment, the method may include comparing atleast one of the one or more properties to a predetermined range for theat least one of the one or more properties. Such an embodiment mayinclude assessing performance of the system based on results of thecomparing step. If the at least one of the one or more properties of theregion is outside of the predetermined range, another such embodiment ofthe method may include determining if one or more corrective stepsshould be performed on the system.

In another embodiment, the method may include comparing one or moreproperties of the optimized classification region to the one or moreproperties of the region. In one such embodiment, the one or moreproperties of the optimized classification region and the region includesize, centroid position, slope of a best-fit line to the values insidethe region or the optimized classification region, offset of thebest-fit line, or some combination thereof. In another such embodiment,if the results of this comparing step exceed a predetermined limit, themethod may include determining if the system is malfunctioning.

In a further embodiment, the optimized classification region includes aportion of the classification space, and a probability that theparticles will have values located in the portion of the classificationspace is greater than a predetermined probability. In anotherembodiment, the optimized classification region excludes a portion ofthe classification space, and a probability that the particles will havevalues located in the portion of the classification space is less than apredetermined probability. Each of the embodiments of the methoddescribed above may include any other step(s) described herein.

Another embodiment relates to a different method for altering one ormore parameters of a measurement system. This method includes analyzinga sample using the system to generate values from classificationchannels of the system for two or more populations of particles in thesample. The method also includes identifying two or more regions in aclassification space. In each of the two or more regions, the values forone of the two or more populations of particles are located. Inaddition, the method includes determining two or more optimizedclassification regions. Each of the two or more optimized classificationregions corresponds to one of the two or more regions. The methodfurther includes interpolating an optimized classification region for anadditional population of particles not included in the sample using thetwo or more optimized classification regions. This embodiment may alsoinclude any other step(s) described herein.

An additional embodiment relates to a computer-implemented method foraltering one or more parameters of a measurement system. Thecomputer-implemented method includes identifying a region in aclassification space in which values for a population of particles of asample are located. The values include values generated byclassification channels of the system during analysis of the sample. Thecomputer-implemented method also includes determining an optimizedclassification region for the population using one or more properties ofthe region. The optimized classification region contains a predeterminedpercentage of the values for the population. The optimizedclassification region is used for classification of particles inadditional samples. This embodiment of the computer-implemented methodmay include any other step(s) described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention will become apparent uponreading the following detailed description and upon reference to theaccompanying drawings in which:

FIG. 1 is a flow chart illustrating one embodiment of a method foraltering one or more parameters of a measurement system;

FIG. 2 is a plot illustrating flow cytometer data with 2-parameterfluorescence data for a single population of dyed particles;

FIG. 3 is a plot illustrating the data of FIG. 2, which is rotated, witha best-fit line;

FIG. 4 is a plot illustrating a 3-standard deviation ellipse boundingthe data of FIG. 3 in rotated coordinates;

FIG. 5 is a plot illustrating an optimized elliptical, classificationregion, the original region of FIG. 2, and values for the population ofparticles;

FIG. 6 is a plot illustrating a larger boundary around the optimizedclassification region of FIG. 5;

FIG. 7 is a plot illustrating flow cytometer data in a 2-parameterfluorescence plot with four distinct regions, each for differentpopulations of particles;

FIG. 8 is a plot illustrating the four original regions of FIG. 7 with anew parametrically generated optimized classification region;

FIG. 9 is a plot illustrating a region enlarged in the CH1 direction;

FIG. 10 is a plot illustrating a region enlarged in the CH1 and CH2directions; and

FIG. 11 is a schematic diagram illustrating one example of a flowcytometer-based measurement system that may be used to carry out themethods described herein.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and will herein be described in detail. Itshould be understood, however, that the drawings and detaileddescription thereto are not intended to limit the invention to theparticular form disclosed, but on the contrary, the intention is tocover all modifications, equivalents and alternatives falling within thespirit and scope of the present invention as defined by the appendedclaims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term “particles” is used herein to generally refer to particles,microspheres, polystyrene beads, microparticles, gold nanoparticles,quantum dots, nanodots, nanoparticles, nanoshells, beads, microbeads,latex particles, latex beads, fluorescent beads, fluorescent particles,colored particles, colored beads, tissue, cells, micro-organisms,organic matter, non-organic matter, or any other discrete substrates orsubstances known in the art. The particles may serve as vehicles formolecular reactions. Examples of appropriate particles are illustratedin U.S. Pat. Nos. 5,736,330 to Fulton, 5,981,180 to Chandler et al.,6,057,107 to Fulton, 6,268,222 to Chandler et al., 6,449,562 to Chandleret al., 6,514,295 to Chandler et al., 6,524,793 to Chandler et al., and6,528,165 to Chandler, which are incorporated by reference as if fullyset forth herein. The measurement systems and methods described hereinmay be used with any of the particles described in these patents. Inaddition, particles for use in flow cytometry such as LabMAPmicrospheres, which may also be referred to as xMAP microspheres, may beobtained commercially from Luminex Corporation. The terms “particles”and “microspheres” are used interchangeably herein.

Methods are described herein for altering one or more parameters of ameasurement system. In particular, the methods can be used to “tune” ameasurement system such that the system can classify particles from aparticular population with increased accuracy and such that theprobability that the system will misclassify particles is decreased. Asused herein, the term “classification” is generally defined asdetermining the identity of individual particles in a sample. Theidentity relates to the population to which individual particles belong.Such classification is of particular importance since often a samplewill be analyzed with multiple, different populations of particles in asingle experiment. For example, the different populations of particlestypically have at least one different characteristic such as the type ofsubstance coupled to the particles and/or the quantity of substancescoupled to the particles such that the presence of different analyteswithin the sample can be detected and/or quantified in a singleexperiment. Therefore, to interpret the measurement results, theidentify or classification of individual particles used in theexperiment are determined such that other measurement values can becorrelated to the properties of the individual particles.

Systems that can be configured to perform the methods described hereininclude, but are not limited to, the Luminex 100, the Luminex HTS, theLuminex 100E, and any further add-ons to this family of products thatare available from Luminex Corporation. One general example of suchsystems is described further herein. In particular, the methodsdescribed herein can use the “Map method” of classifying particles in aflow cytometer-based system to alter one or more parameters of the flowcytometer-based system. However, it is to be understood that the methodsdescribed herein may be used to alter one or more parameters of anymeasurement system that is configured to determine the identity orclassification of individual particles or other discrete substances. Oneexample of such a measurement system is a fluorescent imaging system. Inaddition, the methods described herein are not limited to use inincreasing the accuracy of particle classification. For example, themethods described herein may be equally applied to determining otherclassification parameters such as, but not limited to, the identity orquantity of a reaction product present on the particles. Furthermore,although various values are described herein that can be used with themethods, it is to be understood that the methods described herein may beused with any measurable parameter of particles that can be used todetermine one or more characteristics of the particles.

As will be evident to one of ordinary skill in the art having thebenefit of the description of the various embodiments provided herein,the methods described herein provide several advantages for analyzingvalues generated by a measurement system. In particular, the methodsdescribed herein provide greater system accuracy than is currentlyavailable without altering the physical parameters of the measurementsystem (e.g., without altering the accumulated tolerance of variouscomponents of the measurement system). Therefore, the methods describedherein provide greater system accuracy without complicating themanufacturing process, increasing the manufacturing time, decreasingmanufacturing throughput, and increasing overall system cost. Inaddition, since the methods described herein allow the classificationspace of a region corresponding to a population of particles to bereduced without decreasing the accuracy of the measurement system, themethods described herein provide greater system-to-system matching thanis currently available. Since the system-to-system matching can beincreased without altering the physical components of the measurementsystem, the methods described herein provide greater system-to-systemmatching without complicating the manufacturing process, increasing themanufacturing time, decreasing manufacturing throughput, and increasingoverall system cost.

FIG. 1 illustrates an embodiment of a method for altering one or moreparameters of a measurement system. It is noted that many of the stepsshown in FIG. 1 are not essential to practice of the method. One or moresteps may be omitted or added to the method illustrated in FIG. 1, andthe method can still be practiced within the scope of this embodiment.In addition, it is to be noted that one or more steps of the method maybe automated. In this manner, one or more steps of the method may beperformed without supervision or instructions from a user of themeasurement system. Alternatively, one or more steps of the method maybe performed based on input from or interaction with a user such as anoperator of the system, a field technician, or manufacturing personnel.Furthermore, one or more steps of the method or even all of the steps ofthe method may be computer-implemented. In this manner, one or more ofthe steps can be performed by a processor of the system, an algorithm orother program instructions that are operable to execute one or more ofthe steps on a processor such as those described herein or any otherhardware or software known in the art.

As shown in step 10 of FIG. 1, the method may optionally includecalibrating the measurement system. The method may include calibratingthe system using normal or standard calibration reagents and procedures.One example of such a calibration procedure is the Map CalibrationProcedure, which is commonly used on flow cytometer based measurementsystems that are commercially available from Luminex Corporation. Forexample, a flow cytometer based measurement system identifies particlesthat pass through the system based on the measured intensity of two ormore dyes internal or otherwise attached to the particles. Thisidentification technique can also be used to identify a calibrationmicrosphere that contains known quantities of fluorescent intensity inall channels (both reporter and classification). After the calibrationmicrosphere measurement is performed, a fine correction factor can beapplied to the reporter and/or classification channels for samplemicrosphere measurements. Additional examples of calibration techniquesthat may be used in step 10 are illustrated in U.S. patent Ser. No.10/918,647 entitled “Methods for Controlling One or More Parameters of aFlow Cytometer Type Measurement System” filed Aug. 12, 2004 by Roth etal., which claims priority to U.S. Pat. No. 60/494,824 entitled “RealTime System Calibration Methods” filed Aug. 13, 2003, by Roth, both ofwhich are incorporated by reference as if fully set forth herein. Anyother calibration technique known in the art can be used in thisoptional calibration step.

The method also includes analyzing a sample to generate values fromclassification channels of the system for a population of particles inthe sample, as shown in step 12. In one embodiment, the sample mayinclude a Map Calibration Reagent. A Map Calibration Reagent includesone or more populations of dyed microspheres from the product group thatis commercially available as the 100-region LabMAP from LuminexCorporation, or possibly populations of microspheres that have similarintensities to those in the 100-region LabMAP, but are not part of theLabMAP. In addition, the sample may include any other appropriate sampleknown in the art such as a sample that includes at least one populationof particles.

In one embodiment, the values from the classification channels includefluorescence values. In a different embodiment, the values from theclassification channels include light scatter intensity values. In otherembodiments, the values from the classification channels include volumemeasurements of the particles. In some embodiments, the values mayinclude some combination of fluorescence values, light scatter intensityvalues, and volume measurements of the particles. Each of thesedifferent types of values may be expressed in linear units orlogarithmic units.

In addition, the method includes identifying a region in aclassification space in which the values for the population are located,as shown in step 14. The values may be organized in the classificationspace using any method known in the art such as a two-dimensional plot,with the values for the different classification channels along each ofthe two axes. Such a plot, in the context of flow cytometry-basedinstruments, is commonly referred to as a “map.” Identifying the regionin the classification space in which the values for the population arelocated may include defining a boundary around a cluster of values thatcontains values from as many of the particles in the population aspossible, but excludes to the degree possible any particles that are notmembers of the population. The boundary, therefore, identifies the areain the classification space belonging to the region. Such a boundary maybe generated by a user or by program instructions.

The method may optionally include determining one or more properties ofthe values of the population located within the region (hereafterreferred to as properties of the region), as shown in step 16. The oneor more properties of the region may include an average, mean, peak, ormedian of the values in the region and a standard deviation of theregion. Such properties of the region may be determined using any methodknown in the art. The one or more properties of the region may be usedto perform a number of functions. For example, in one embodiment, themethod may include comparing at least one of the properties of theregion to a predetermined range for the at least one property, as shownin step 18. The predetermined range may, in some embodiments, berepresentative of typical, acceptable, or expected performance of thesystem. In this manner, the method may optionally include assessing theperformance of the system, as shown in step 20, based on resultsobtained in the comparing step. In other words, if one or moreproperties of the region are significantly different from the expectedvalues for the properties, these significant differences may be detectedby the comparing step and can be used to monitor system performance. Inanother example, if at least one of the properties is outside of thepredetermined range, the method may include determining if one or morecorrective steps should be performed on the system, as shown in optionalstep 22. The corrective step(s) may include, for example, calibration,maintenance, repair, troubleshooting, rebooting, etc.

The method includes determining an optimized classification region forthe population using the one or more properties of the region, as shownin step 24. The optimized classification region contains a predeterminedpercentage of the values for the population. The predeterminedpercentage may, in some embodiments, reflect the desired accuracy of themeasurement system (e.g., 98%, 95%, 90%, etc.). In another embodiment,the predetermined percentage may be determined based on thecharacteristics of the values in the region such as the distribution ofthe values within the region and other properties of the regiondescribed above. Since the optimized classification region will includeonly a predetermined percentage of the values in the region, theoptimized classification region will usually, but not necessarilyalways, have a size in the classification region that is less than asize of the region in the classification space. In general, theoptimized classification region will have one or more properties thatare different from the one or more properties of the region. The one ormore different properties may include size, shape, position, or somecombination thereof. In one embodiment, the size of the optimizedclassification region may be a minimum size that contains thepredetermined percentage of the values for the population. In someembodiments, the optimized classification region centers on a median ofthe values in the region plus a number of standard deviations (forexample, two or three) away from the median. The median of the valuesmay be determined empirically in one example. In another embodiment, theoptimized classification region is defined by a predetermined sizedboundary surrounding a median of the values for the population.

The optimized classification region can be used for classification ofparticles in additional samples. In particular, values of theclassification channels generated for particles of other samples may becompared to this and possibly other optimized classification regions. Ifthe values for a particle fall within one of the optimizedclassification regions, the particle is assigned the classificationassociated with that optimized classification region.

The optimized classification region provides significant advantages overother methods for classification such as greater system accuracy inclassifying particles and greater system-to-system matching for particleclassification. For example, the optimized classification regionincludes a portion of the classification space. The portion of theclassification space within the optimized classification region may bedetermined as described above such that a probability that the particleswill have values located in the portion of the classification space isgreater than a predetermined probability. The predetermined probabilitymay be determined based on a number of parameters such as statistical orother mathematical analysis of the expected results of particlemeasurements. The optimized classification region also excludes aportion of the classification space. The portion of the classificationspace excluded from the optimized classification region may bedetermined such that a probability that the particles will have valueslocated in the excluded portion of the classification space is less thana predetermined probability. This predetermined probability will bedifferent than the one described above, but both probabilities may bedetermined in the same manner.

In some embodiments, the method may include determining one or moreproperties of the optimized classification region, as shown in optionalstep 26. However, it is to be noted that the optimized classificationregion can be used for classification of particles in additional sampleseven if the one or more properties are not determined for the optimizedclassification region. The one or more properties of the optimizedclassification region may be used to perform a number of additionalsteps. For example, the method may include comparing one or moreproperties of the optimized classification region to the one or moreproperties of the region, as shown in optional step 28. The one or moreproperties of the optimized classification region and the region thatare compared may include, but are not limited to, size, centroidposition, slope of a best-fit line to the values inside the region orthe optimized classification region, offset of the best-fit line, orsome combination thereof. The results of the comparison step may also beused to perform one or more additional steps. In one such example, ifthe results of the comparing step exceed a predetermined limit, themethod may include determining if the system is malfunctioning, as shownin optional step 30. The predetermined limit may be based on typical,acceptable, or expected properties for the regions. If it is determinedthat the system is malfunctioning, the method may include determining ifone or more corrective steps such as those described above should beperformed on the system.

The sample that is analyzed in step 12 may include one or moreadditional populations of particles. In one embodiment, the method maybe performed for the one or more additional populations, as shown inoptional step 32. In this manner, the method may include determining anoptimized classification region for each of the one or more additionalpopulations of particles. An optimized classification region may bedetermined for additional populations as described herein. It is to benoted that an optimized classification region may optionally not bedetermined for every population of particles in the sample. When two ormore optimized classification regions are determined for a sample, themethod may include interpolating an optimized classification region foranother population of particles that was not included in the sample, asshown in optional step 34, using the two or more optimizedclassification regions. In addition, optimized classification regionsfor more than one population of particles not included in the sample maybe determined in this manner.

Various steps illustrated in FIG. 1 may be combined to generatedifferent embodiments of a method for altering one or more parameters ofa measurement system. For example, one embodiment may include analyzinga sample using the system to generate values from classificationchannels of the system for two or more populations of particles in thesample, which may be performed as described with respect to step 12. Inaddition, this method includes identifying two or more regions in aclassification space, which may be performed as described with respectto step 14. In each of the two or more regions, the values for one ofthe two or more populations of particles are located. The method alsoincludes determining two or more optimized classification regions, whichmay be performed as described with respect to step 32. Each of the twoor more optimized classification regions corresponds to one of the twoor more regions. The method further includes interpolating an optimizedclassification region for an additional population of particles notincluded in the sample using the two or more optimized classificationregions, which may be performed as described with respect to step 34.This embodiment of the method may include any other step(s) describedherein.

In another example, an embodiment of a computer-implemented method foraltering one or more parameters of a measurement system includesidentifying a region in a classification space in which values for apopulation of particles of a sample are located, which may be performedas described with respect to step 14. The values include valuesgenerated by classification channels of the system during analysis ofthe system. This computer-implemented method also includes determiningan optimized classification region for the population using one or moreproperties of the region, which may be performed as described withrespect to step 24. As described further herein, the optimizedclassification region contains a predetermined percentage of the valuesfor the population. The optimized classification region can be used forclassification of particles in additional samples as described herein.This embodiment of a computer-implemented method may include any otherstep(s) described herein.

One example of determining an optimized classification region mayinclude the following steps, which may produce data as illustrated inFIGS. 2-6. It is noted that the data illustrated in FIGS. 2-6 is notmeant to be exemplary or limiting examples of the methods describedherein. Instead, this data is presented merely to enhance understandingof the methods described herein. FIG. 2 illustrates data obtained from aflow cytometer-like device in which two colors of fluorescence are usedto identify the particles. The two colors of fluorescence were detectedusing two different classification channels, CH1 and CH2. A boundary isdrawn around the cluster of data values that contains fluorescence datafrom as many of the particles in the population as possible, butexcludes to the degree possible any particles that are not members ofthe population. The white region around the data points is bounded by ahand-drawn boundary, which adequately includes values for thepopulation, but also includes extra space in which members of thepopulation are unlikely to exhibit fluorescence. This region can beoptimized by eliminating some of this extra space, so that in situationswhere the sample contains other fluorescent particles that are notmembers of this population, they can be easily discriminated by theoptimized classification region.

A linear regression is applied to the data shown in FIG. 2 to obtain theslope of the line that best fits this population. A “population” canthen be defined as including any data within, for example, 25% of themedian CH1 and CH2 values (i.e., a box bounded on the sides byMedian_CH1+25%, Median_CH1−25%, and on the top and bottom by MedianCH2+25%, Median_CH2−25%). After determining this slope, the entire dataset is rotated so that the best-fit line is essentially horizontal (asshown in FIG. 3), with new coordinates of CH1′ and CH2′. Using therotated data set, a median and standard deviation is determined for thepopulation in the CH1′ and CH2′ dimensions.

As shown in FIG. 4, an ellipse is created having a center that is themedian values in CH1′ and CH2′ and whose major and minor axes (althoughnot necessarily in that order) are some constant C times the standarddeviations in CH1′ and CH2′. The constant is adjusted until the ellipsecontains the desired percentage of particles in the population. In thisexample, giving C a value of 3 creates a boundary that encircles 98.9%of the population (as shown in FIG. 4). Finally, the set of pointswithin the ellipse are rotated back to the original dimensions of CH1and CH2, as shown in FIG. 5, and the resulting ellipse in the originaldimensions is the optimized classification region. As further shown inFIG. 5, the optimized classification region has a size that is muchsmaller than the original classification region.

Additionally, a larger boundary could be created around the region bygenerating an ellipse with axes corresponding to a higher number ofstandard deviations than the classification region. If no otherclassification regions (for other populations) are placed within thislarger boundary, no more than a predetermined percentage of theparticles from this region will erroneously be classified in the otherregion. The converse is also true, given that a similar boundary hasalso been generated for the other population of particles. In thisexample, the classification boundary is 3 standard deviations away fromthe centroid of the population in both axes. The larger boundary mightbe 5 standard deviations away, which in this case would enclose 100% ofthe 96 particles in the population. FIG. 6 shows the original region(white area), the population of data points, the first optimizedclassification region boundary defined by the smaller boundary (smallerellipse), and the optimized classification region defined by the largerboundary based on 5 standard deviations in both of the rotatedcoordinate axes (larger ellipse).

One example of optimizing two or more regions may include the followingsteps, which may produce data as illustrated in FIGS. 7-8. It is notedthat the data illustrated in FIGS. 7-8 is not meant to be exemplary orlimiting examples of the methods described herein. Instead, this data ispresented merely to enhance understanding of the methods describedherein. FIG. 7 shows data generated by a flow cytometer-like deviceduring analysis of a sample containing four different populations offluorescently-dyed particles. The optimized elliptical regions have beencreated for each region corresponding to one of the populations asdescribed in the above example, but ellipses with major and minor axesof 5 standard deviations were used so that the optimized classificationregions appear distinct from the data points. In practice, smallerclassification regions would probably be optimal.

These four optimized classification regions can be used to generate anadditional optimized classification region for another populationwithout actually having data for the new population. The parameters usedto generate the additional optimized classification region are itslocation in terms of CH1 and CH2 coordinates. A model is generated fromthe four optimized classification regions that were created from actualdata values generated by analysis of the sample, and input parametersfor the model are also region locations in CH1 and CH2 coordinates.Then, the additional optimized classification region can be interpolatedanywhere in the CH1-CH2 space without actually having measurement datafor the particular optimized classification region being generated.

For the four regions shown in FIG. 7, the region coordinates are shownin Table 1:

TABLE 1 CH1 CH2 Region 1 120 118 Region 2 176 145 Region 3 232 197Region 4 134 212These coordinates represent the positions of the centroids of theoriginal classification region (as shown by the white areas in FIG. 7).

Based on the coordinates of the positions of the centroids, thefollowing statistics are calculated for each region:

-   -   1. Centroid shift in CH1 (the vector connecting the original        region centroid in CH1 and the optimized classification region        centroid in CH1);    -   2. Centroid shift in CH2 (the vector connecting the original        region centroid in CH2 and the optimized classification region        centroid in CH2);    -   3. Slope of best-fit line through values for a population;    -   4. Standard deviations of the values for a population in CH1′        and CH2′ (rotated) coordinates; and    -   5. Number of standard deviations required to encircle the        desired percentage of values for particles in each population.        A regression model is built to create models for each of these        five statistics as functions of the CH1 and CH2 coordinates        shown in Table 1. For example, with this data, the form of the        expression for statistic 3 was the following equation:

Slope=0.515+(CH1*0.00205)+(CH2*0.00165)

Once these five statistics are calculated for any pair of coordinates inCH1 and CH2, an optimized elliptical region can be automaticallygenerated using only that pair of numbers. FIG. 8 shows the originalfour regions from FIG. 7 plus a fifth optimized elliptical regiongenerated for the original region labeled with the arrow. This processcould be repeated multiple times, so that many new optimized regionscould be generated using only the data collected from the original fourpopulations, as shown in FIG. 7.

One example of enlarging a particular region to account for extraneoussources of error may include the following steps, which may produce dataas illustrated in FIGS. 9-10. It is noted that the data illustrated inFIGS. 9-10 is not meant to be exemplary or limiting examples of themethods described herein. Instead, this data is presented merely toenhance understanding of the methods described herein. Experienceindicates that other sources of error, such as aged calibrationreagents, might increase the size of the boundary that captures thedesired percentage of particles in each region (which may be performedas described in the above examples). If it is known that the centroid ofa particular region may deviate by a certain number of units based onthese sources of error, then the region may be widened by that amount.In the example of determining an optimized classification regiondescribed above, if the user wants to allow for a shift of 2 counts ineither direction for the CH1 axis, then the boundary to the right andleft of the region's centroid in the CH1 direction can both be shiftedaway from the centroid by that amount (as shown in FIG. 9). Similarly,the method could be applied to the CH2 direction, or to both directionsfor the same region (as shown in FIG. 10).

FIG. 11 illustrates one example of a measurement system that may be usedto perform the methods described herein. It is noted that FIG. 11 is notdrawn to scale. In particular, the scale of some of the elements of thefigure are greatly exaggerated to emphasize characteristics of theelements. Some elements of the measurement system have not been includedin the figure for the sake of clarity.

In FIG. 11, the measurement system is shown along a plane through thecross-section of cuvette 40 through which microspheres 42 flow. In oneexample, the cuvette may be a standard quartz cuvette such as that usedin standard flow cytometers. Any other suitable type of viewing ordelivery chamber, however, may also be used to deliver the sample foranalysis. The measurement system includes light source 44. Light source44 may include any appropriate light source known in the art such as alaser. The light source may be configured to emit light having one ormore wavelengths such as blue light or green light. Light source 44 maybe configured to illuminate the microspheres as they flow through thecuvette. The illumination may cause the microspheres to emit fluorescentlight having one or more wavelengths or wavelength bands. In someembodiments, the system may include one or more lenses (not shown)configured to focus light from the light source onto the microspheres orthe flowpath. The system may also include more than one light source. Inone embodiment, the light sources may be configured to illuminate themicrospheres with light having different wavelengths or wavelength bands(e.g., blue light and green light). In some embodiments, the lightsources may be configured to illuminate the microspheres at differentdirections.

Light scattered forwardly from the microspheres may be directed todetection system 46 by folding mirror 48 or another such light directingcomponent. Alternatively, detection system 46 may be placed directly inthe path of the forwardly scattered light. In this manner, the foldingmirror or other light directing components may not be included in thesystem. In one embodiment, the forwardly scattered light may be lightscattered by the microspheres at an angle of about 180 degrees from thedirection of illumination by light source 44, as shown in FIG. 11. Theangle of the forwardly scattered light may not be exactly 180 degreesfrom the direction of illumination such that incident light from thelight source may not impinge upon the photosensitive surface of thedetection system. For example, the forwardly scattered light may belight scattered by the microspheres at angles less than or greater than180 degrees from the direction of illumination (e.g., light scattered atan angle of about 170 degrees, about 175 degrees, about 185 degrees, orabout 190 degrees).

Light scattered by the microspheres at an angle of about 90 degrees fromthe direction of illumination may also be collected. In one embodiment,this scattered light may be separated into more than one beam of lightby one or more beamsplitters or dichroic mirrors. For example, lightscattered at an angle of about 90 degrees to the direction ofillumination may be separated into two different beams of light bybeamsplitter 50. The two different beams of light may be separated againby beamsplitters 52 and 54 to produce four different beams of light.Each of the beams of light may be directed to a different detectionsystem, which may include one or more detectors. For example, one of thefour beams of light may be directed to detection system 56. Detectionsystem 56 may be configured to detect light scattered by themicrospheres.

Scattered light detected by detection system 46 and/or detection system56 may generally be proportional to the volume of the particles that areilluminated by the light source. Therefore, output signals of detectionsystem 46 and/or output signals of detection system 46 may be used todetermine a diameter and/or volume of the particles that are in theillumination zone or detection window. In addition, the output signalsof detection system 46 and/or detection system 56 may be used toidentify more than one particle that are stuck together or that arepassing through the illumination zone at approximately the same time.Therefore, such particles may be distinguished from other samplemicrospheres and calibration microspheres. Furthermore, the outputsignals of detection system 46 and/or detection system 56 may be used todistinguish between sample microspheres and calibration microspheres.

The other three beams of light may be directed to detection systems 58,60, and 62. Detection systems 58, 60, and 62 may be configured to detectfluorescence emitted by the microspheres. Each of the detection systemsmay be configured to detect fluorescence of a different wavelength or adifferent range of wavelengths. For example, one of the detectionsystems may be configured to detect green fluorescence. Another of thedetection systems may be configured to detect yellow-orangefluorescence, and the other detection system may be configured to detectred fluorescence.

In some embodiments, spectral filters 64, 66, and 68 may be coupled todetection systems 58, 60, and 62, respectively. The spectral filters maybe configured to block fluorescence of wavelengths other than that whichthe detection systems are configured to detect. In addition, one or morelenses (not shown) may be optically coupled to each of the detectionsystems. The lenses may be configured to focus the scattered light oremitted fluorescence onto a photosensitive surface of the detectors.

Each of the detector's output currents is proportional to thefluorescent light impinging on it and results in a current pulse. Thecurrent pulse may be converted to a voltage pulse, low pass filtered,and then digitized by an A/D converter. The conversion, filtering, anddigitizing may be performed using any suitable components known in theart. The measurement system may also include processor 70. Processor 70may be coupled to the detectors by one or more transmission media andoptionally one or more components interposed between the processor andthe detectors. For example, processor 70 may be coupled to detectionsystem 56 by transmission medium 72. The transmission medium may includeany suitable transmission medium known in the art and may include“wired” and “wireless” portions. The processor may include, in oneexample, a DSP that is configured to integrate the area under the pulseto provide a number which represents the magnitude of the fluorescence.The processor may also be configured to perform one or more of the stepsof the embodiments described herein.

In some embodiments, the output signals generated from fluorescenceemitted by the microspheres may be used to determine an identity of themicrospheres and information about a reaction taking place on thesurface of the microspheres. For example, output signals of two of thedetection systems may be used to determine an identity of themicrospheres, and output signals of the other detection system may beused to determine a reaction taking place on the surface of themicrospheres. Therefore, the selection of the detectors and the spectralfilters may vary depending on the type of dyes incorporated into orbound to the microspheres and/or the reaction being measured (i.e., thedye(s) incorporated into or bound to the reactants involved in thereaction).

The values generated by detections systems 46, 56, 58, 60, and 62 may beused in the methods described herein. In one particular example, thevalues generated by two of the detection systems for a microsphere maybe compared, in a classification space, to one or more optimizedclassification regions, which are determined as described above. Theclassification of the microsphere may be determined as theclassification assigned to the optimized classification region in whichthe values are located.

The detection systems that are used to determine an identity of thesample microspheres (e.g., detection systems 58 and 60) may be avalanchephotodiodes (APDs), a photomultiplier tube (PMT), or anotherphotodetector. The detection system that is used to identify a reactiontaking place of the surface of the microspheres (e.g., detection system62) may be a PMT, an APD, or another form of photodetector.

Although the system of FIG. 11 is shown to include two detection systemshaving two different detection windows for distinguishing betweenmicrospheres having different dye characteristics, it is to beunderstood that the system may include more than two such detectionwindows (i.e., 3 detection windows, 4 detection windows, etc.). In suchembodiments, the system may include additional beamsplitters andadditional detection systems having other detection windows. Inaddition, spectral filters and/or lenses may be coupled to each of theadditional detection systems. In another embodiment, the system mayinclude two or more detection systems configured to distinguish betweendifferent materials that are reacted on the surface of the microspheres.The different reactant materials may have dye characteristics that aredifferent than the dye characteristics of the microspheres.

Additional examples of measurement systems that may be used to performthe methods described herein are illustrated in U.S. Pat. Nos. 5,981,180to Chandler et al., 6,046,807 to Chandler, 6,139,800 to Chandler,6,366,354 to Chandler, 6,411,904 to Chandler, 6,449,562 to Chandler etal., and 6,524,793 to Chandler et al., which are incorporated byreference as if fully set forth herein. The measurement system describedherein may also be further configured as described in these patents.

Program instructions implementing methods such as those described hereinmay be transmitted over or stored on a carrier medium. The carriermedium may be a transmission medium such as a wire, cable, or wirelesstransmission link, or a signal traveling along such a wire, cable, orlink. The carrier medium may also be a storage medium such as aread-only memory, a random access memory, a magnetic or optical disk, ora magnetic tape.

In an embodiment, a processor may be configured to execute the programinstructions to perform a computer-implemented method according to theabove embodiments. The processor may take various forms, including apersonal computer system, mainframe computer system, workstation,network appliance, Internet appliance, personal digital assistant(“PDA”), television system or other device. In general, the term“computer system” may be broadly defined to encompass any device havingone or more processors, which executes instructions from a memorymedium.

The program instructions may be implemented in any of various ways,including procedure-based techniques, component-based techniques, and/orobject-oriented techniques, among others. For example, the programinstructions may be implemented using ActiveX controls, C++ objects,JavaBeans, Microsoft Foundation Classes (“MFC”), or other technologiesor methodologies, as desired.

It will be appreciated to those skilled in the art having the benefit ofthis disclosure that this invention is believed to provide methods foraltering one or more parameters of a measurement system. Furthermodifications and alternative embodiments of various aspects of theinvention will be apparent to those skilled in the art in view of thisdescription. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1. A method for altering one or more parameters of a measurement system,comprising: analyzing a sample using the system to generate values fromclassification channels of the system for two or more populations ofparticles in the sample; identifying two or more regions in aclassification space, wherein in each of the two or more regions thevalues for one of the two or more populations of particles are located;determining two or more optimized classification regions, wherein eachof the two or more optimized classification regions corresponds to oneof the two or more regions; and interpolating an optimizedclassification region for an additional population of particles notincluded in the sample using the two or more optimized classificationregions.
 2. The method of claim 1, wherein the step of interpolating theoptimized classification region for the additional population ofparticles comprises interpolating optimized classification regions for aplurality of different populations of particles not included in thesample using the two of more optimized classification regions.
 3. Themethod of claim 1, wherein the step of interpolating the optimizedclassification region for the additional population of particlescomprises: calculating one or more statistics for each of the two ormore optimized classification regions; creating a regression model foreach of one or more statistics as a function of centroid coordinates ofthe two or more optimized classification regions; inputting differentcentroid coordinates associated with the additional population ofparticles into the regression model to compute one or more statisticsfor the optimized classification region for the additional population ofparticles; and generating the optimized classification region for theadditional population of particles based on said computed statistics. 4.A method for altering one or more parameters of a measurement system,comprising: analyzing a sample using the system to generate values fromclassification channels of the system for a population of particles inthe sample; identifying a region in a classification space in which thevalues for the population are located; and determining an optimizedclassification region for the population using one or more properties ofthe values of the population, wherein the optimized classificationregion comprises a portion of the classification space, wherein aprobability that the particles will have values located in the portionof the classification space is greater than a predetermined probability,and wherein the optimized classification region is used forclassification of particles in additional samples.
 5. The method ofclaim 4, wherein the optimized classification region has one or moreproperties that are different from one or more respective properties ofthe identified region, and wherein the one or more properties comprisesize, shape, position, or some combination thereof.
 6. The method ofclaim 4, wherein the one or more properties of the values of thepopulation comprise an average, mean, peak, or median of the values ofthe population and a standard deviation of the values of the population.7. The method of claim 4, wherein the optimized classification regioncenters on a median of the values for the population plus a number ofstandard deviations away from the median.
 8. The method of claim 4,wherein the optimized classification region is defined by apredetermined sized boundary surrounding a median of the values for thepopulation.
 9. The method of claim 4, wherein the sample comprises oneor more additional populations of particles, the method furthercomprising performing the method for the one or more additionalpopulations.
 10. The method of claim 4, wherein the sample comprises oneor more additional populations of particles, the method furthercomprising performing the method for the one or more additionalpopulations and interpolating an optimized classification region foranother population of particles that was not included in the sampleusing the optimized classification regions of the population and the oneor more additional populations.
 11. The method of claim 4, wherein theone or more properties of the values of the population comprise anaverage, mean, peak, or median of the values of the population and astandard deviation of the values of the population, the method furthercomprising comparing at least one of the one or more properties of thevalues of the population to a predetermined range for the at least oneof the one or more properties of the values of the population.
 12. Themethod of claim 11, further comprising assessing performance of thesystem based on results of said comparing.
 13. The method of claim 11,further comprising determining if one or more corrective steps should beperformed on the system upon detecting the at least one of the one ormore properties is outside of the predetermined range.
 14. The method ofclaim 4, further comprising comparing one or more properties of theoptimized classification region to the one or more properties of theidentified region.
 15. The method of claim 14, wherein the one or moreproperties of the optimized classification region and the identifiedregion comprise size, centroid position, slope of a best-fit line to thevalues inside the identified region or the optimized classificationregion, offset of the best-fit line, or some combination thereof. 16.The method of claim 14, further comprising determining if the system ismalfunctioning upon detecting one or more properties of the optimizedclassification region exceed one or more respective properties of theidentified region.
 17. A method for altering one or more parameters of ameasurement system, comprising: identifying a region in a classificationspace in which values for a population of particles of a samplecorrespond, wherein the values comprise values generated byclassification channels of the system during analysis of the sample; anddetermining an optimized classification region for the population usingone or more properties of the values of the population, wherein theoptimized classification region is used for classification of particlesin additional samples, and wherein the program instructions fordetermining the optimized classification region comprise programinstructions for: creating a provisional region comprising: a centerthat is statistically representative of the values for the population;and one or more dimensions which are characterized by a firstpredetermined constant multiplied by standard deviations of the valuesfor the population; determining a percentage of particles arrangedwithin the provisional region; and performing one or more responsiveactions based upon the step of determining the percentage of particles,wherein the responsive actions comprise: assigning the provisionalregion as the optimized classification region upon determining thepercentage of particles is greater than or equal to the presetpercentage; adjusting the one or more dimensions of the provisionalregion upon determining the percentage of particles is less than thepreset percentage, wherein the step of adjusting the one or moredimensions comprises multiplying the standard deviations of the valuesfor the population by a different predetermined constant; andreiterating the steps of determining the percentage of particles andperforming the one or more responsive actions subsequent to the step ofadjusting the axes until a provisional region is formed having apercentage of the particles greater than or equal to the predeterminedpercentage.
 18. The method of claim 17, further comprising comparing oneor more properties of the optimized classification region to the one ormore properties of the identified region.
 19. The method of claim 18,further comprising assessing performance of the system based on resultsof said comparing.
 20. The method of claim 18, further comprisingdetermining if the system is malfunctioning upon detecting one or moreproperties of the optimized classification region exceed one or morerespective properties of the identified region.